Fusion protein useful for vaccination against rotavirus

Abstract

The present invention relates to recombinantly constructed polypeptides useful for preparing vaccines, in particular for reducing one or more clinical signs caused by a rotavirus infection. More particular, the present invention is directed to a fusion protein comprising in N- to C-terminal direction (i) an immunogenic fragment of a rotavirus VP8 protein and (ii) an immunoglobulin Fc fragment such as, for example, an IgG Fc fragment, wherein said fusion protein is usable in a method of reducing one or more clinical signs, mortality or fecal shedding caused by a rotavirus infection in swine.

1 . A polypeptide comprising
 an immunogenic fragment of a rotavirus VP8 protein, and   an immunoglobulin Fc fragment.   
     
     
         2 . The polypeptide of  claim 1 , wherein said immunoglobulin Fc fragment is linked to the C-terminus of said immunogenic fragment of a rotavirus VP8 protein via a linker moiety,
 or wherein said immunoglobulin Fc fragment is linked to the C-terminus of said immunogenic fragment of a rotavirus VP8 protein via a peptide bond between the N-terminal amino acid residue of said immunoglobulin Fc fragment and the C-terminal amino acid residue of said immunogenic fragment of a rotavirus VP8 protein.   
     
     
         3 . A polypeptide, in particular the polypeptide of  claim 1  or  2 , wherein said polypeptide is a fusion protein of the formula x-y-z, wherein
 x consists of an immunogenic fragment of a rotavirus VP8 protein; 
 y is a linker moiety; and 
 z is an immunoglobulin Fc fragment. 
 
     
     
         4 . The polypeptide of any one of  claims 1  to  3 , wherein said rotavirus is porcine rotavirus,
 and/or wherein said rotavirus is selected from the group consisting of rotavirus A and rotavirus C. 
 
     
     
         5 . The polypeptide of any one of  claims 1  to  4 , wherein said immunogenic fragment of a rotavirus VP8 protein is an N-terminally extended lectin-like domain of a rotavirus VP8 protein, wherein the N-terminal extension is 1 to 20 amino acid residues, preferably 5 to 15 amino acid residues, in length. 
     
     
         6 . The polypeptide of any one of  claims 1  to  5 , wherein said rotavirus is selected from the group consisting of genotype P[7] rotavirus, genotype P[6] rotavirus and genotype P[13] rotavirus. 
     
     
         7 . The polypeptide of any one of  claims 1  to  6 , wherein the immunogenic fragment of a rotavirus VP8 protein consists of or is a consensus sequence of a portion of a rotavirus VP8 protein, in particular of a portion of a rotavirus A VP8 protein,
 and wherein said consensus sequence of a portion of a rotavirus VP8 protein is preferably obtainable by a method comprising the steps of:
 translating a plurality of nucleotide sequences encoding a portion of a rotavirus VP8 protein into amino acid sequences, 
 aligning said amino acid sequences to known rotavirus VP8 proteins, preferably by using MUSCLE sequence alignment software UPGMB clustering and default gap penalty parameters, 
 subjecting said aligned sequences to a phylogenetic analysis and generating a neighbor joining phylogeny reconstruction based on rotavirus VP8 protein sequence, in particular importing said aligned amino acid sequences into MEGA7 software for phylogenetic analysis and generating a neighbor joining phylogeny reconstruction based on rotavirus VP8 protein sequence, 
 computing the optimal tree using the Poisson correction method with bootstrap test of phylogeny (n=100), 
 drawing the optimal tree to scale with branch lengths equal to evolutionary distances in units of amino acid substitutions per site over 170 total positions, 
 considering nodes where bootstrap cluster association is greater than 70% as significant, 
 designating nodes with approximately 10% distance and bootstrap cluster associations greater than 70% as clusters, and 
 selecting a cluster and generating the consensus sequences by identifying the greatest frequency per aligned position within the cluster, 
 and optionally, in cases where equivalent proportions of amino acids are observed in an aligned position, selecting the amino acid residue based on reported epidemiological data in conjunction with a predefined product protection profile. 
 
 
     
     
         8 . The polypeptide of any one of  claims 1  to  7 , wherein the immunogenic fragment of a rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or still more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 
     
     
         9 . The polypeptide of any one of  claims 1  to  8 , wherein said immunoglobulin Fc fragment is an immunoglobulin Fc fragment encoded by the genome of a species whose intestinal cells are susceptible to an infection by the rotavirus from which the immunogenic fragment of a rotavirus VP8 protein is derived,
 and/or wherein said immunoglobulin Fc fragment is preferably a swine IgG Fc fragment, 
 and/or wherein said immunoglobulin Fc fragment comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8. 
 
     
     
         10 . The polypeptide of any one of  claims 1  to  9 , wherein said linker moiety is an amino acid sequence being 1 to 50 amino acid residues in length,
 and/or wherein said linker moiety comprises or consists of an amino acid sequence having at least 66%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. 
 
     
     
         11 . The polypeptide of any one of  claims 2  to  10 , wherein said polypeptide comprises a further immunogenic fragment of a rotavirus VP8 protein linked to the C-terminus of said immunoglobulin Fc fragment, wherein said further immunogenic fragment of a rotavirus VP8 protein is preferably linked to the C-terminus of said immunoglobulin Fc fragment via a linker moiety, wherein said linker moiety is in particular a linker moiety as specified in  claim 10 ,
 or wherein said further immunogenic fragment of a rotavirus VP8 protein is linked to the C-terminus of said immunoglobulin Fc fragment via a peptide bond between the N-terminal amino acid residue of said further immunogenic fragment of a rotavirus VP8 protein and the C-terminal amino acid residue of said immunoglobulin Fc fragment, 
 and wherein said further immunogenic fragment of a rotavirus VP8 protein preferably comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or still more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2 to 6, 
 and/or wherein said further immunogenic fragment of a rotavirus VP8 protein is preferably different from the immunogenic fragment of a rotavirus VP8 protein of which the C-terminus is linked to said immunoglobulin Fc fragment. 
 
     
     
         12 . The polypeptide of any one of  claims 1  to  11 , wherein said polypeptide is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16. 
     
     
         13 . A multimer comprising or composed of a plurality of the polypeptide of any one of  claims 1  to  12 , and wherein said multimer is preferably a homodimer formed by a polypeptide of any one of  claims 1  to  12  with a second identical polypeptide. 
     
     
         14 . An immunogenic composition comprising the polypeptide of any one of  claims 1  to  12  and/or the multimer of  claim 13 . 
     
     
         15 . A polynucleotide comprising a nucleotide sequence which encodes the polypeptide of any one of  claims 1  to  12 , and wherein said polynucleotide preferably comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21. 
     
     
         16 . The polypeptide of any one of  claims 1  to  12  or the immunogenic composition of  claim 14  for use as a medicament, preferably for use as a vaccine. 
     
     
         17 . The polypeptide of any one of  claims 1  to  12  or the immunogenic composition of  claim 14  for use in a method of reducing or preventing one or more clinical signs, mortality or fecal shedding caused by a rotavirus infection in a subject or for use in a method of treating or preventing an infection with rotavirus in a subject or for use in a method of treating or preventing an infection with rotavirus in a subject,
 and/or for use in a method for inducing an immune response against rotavirus in a subject. 
 
     
     
         18 . A method of reducing or preventing one or more clinical signs, mortality or fecal shedding caused by a rotavirus infection in a piglet, wherein said method comprises
 administering the polypeptide of any one of  claims 1  to  12  or the immunogenic composition of  claim 14  to a sow, and   allowing said piglet to be suckled by said sow.   
     
     
         19 . The polypeptide or the immunogenic composition according to  claim 17 , or the method of  claim 18 , wherein said one or more clinical signs are selected from the group consisting of
 diarrhea,   rotavirus colonization, in particular rotavirus colonization of the intestine,   lesions, in particular macroscopic lesions,   decreased average daily weight gain, and   gastroenteritis.   
     
     
         20 . The polypeptide or the immunogenic composition according to  claim 17  or  19 , or the method of  claim 18  or  19 , wherein
 said rotavirus infection is an infection with genotype P[23] rotavirus and/or genotype P[7] rotavirus, 
 said infection with a rotavirus is an infection with a genotype P[23] rotavirus and/or genotype P[7] rotavirus, or 
 said immune response against rotavirus is an immune response against genotype P[23] rotavirus and/or genotype P[7] rotavirus. 
 
     
     
         21 . The polypeptide or the immunogenic composition according to  claim 20 , or the method of  claim 20 ,
 wherein said polypeptide comprises an immunogenic fragment of a genotype P[7] rotavirus VP 8 protein, or wherein said immunogenic composition comprises a polypeptide comprising an immunogenic fragment of a genotype P[7] rotavirus VP8 protein,   and wherein preferably said immunogenic fragment of a genotype P[7] rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or still more preferably at least 99% sequence identity with the sequence of SEQ ID NO:3.   
     
     
         22 . A method of producing the immunogenic composition of  claim 14 , wherein the method comprises the steps of:
 (a) permitting infection of susceptible cells in culture with a vector comprising a nucleic acid sequence encoding a polypeptide of any one of  claims 1  to  12 , wherein said polypeptide is expressed by said vector;   (b) thereafter recovering said polypeptide, in particular in the cell culture supernatant, wherein preferably cell debris is separated from said polypeptide via a separation step, preferably including a micro filtration through at least one filter, preferably two filters, wherein the at least one filter preferably has a pore size of about 1 to about 20 μm and/or about 0.1 μm to about 4 μm;   (c) inactivating the vector by adding binary ethylenimine (BEI) to the mixture of step (b);   (d) neutralizing the BEI by adding sodium thiosulfate to the mixture resulting from step (c); and   (e) concentrating the polypeptide in the mixture resulting from step (d) by removing a portion of the liquid from the mixture by a filtration step utilizing a filter with a filter membrane having a molecular weight cut off of between about 5 kDa and about 100 kDa, preferably between about 10 kDa and about 50 kDa;   (f) and optionally admixing the mixture remaining after step (e) with a further component selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof.